Headache disorders, such as migraine, carry an enormous burden for individuals and societies due to their high prevalence, significant disability, and considerable economic costs. Current treatment options for recurrent headaches such as migraine remain inadequate, and development of novel therapies is severely hindered by a poor understanding of the underlying neural mechanisms. A large body of indirect evidence points to the trigeminal sensory innervation of the intracranial meninges and their related blood vessels as key players in the genesis of migraine headaches. Nevertheless, the specific processes that activate these meningeal afferents and thereby drive headaches remain unclear. Current knowledge about the response properties of intracranial meningeal afferents in vivo has been almost exclusively derived from acute single- unit recordings of dural afferents from their trigeminal somata in anesthetized rats with exposed and depressurized meninges. However, these recordings only provide coarse intuition as to the many kinds of natural stimuli that might activate the diversity of meningeal afferents. To overcome these limitations, we propose a novel approach to monitor the activity of meningeal afferents and their unique responses to a large set of complex physiological stimuli, in the intact intracranial space of awake, behaving mice over hours and days. Specifically, we will harness recent developments in genetically encoded calcium indicators together with two-photon microscopy to develop a novel method to study the activity of dozens of meningeal afferents simultaneously in awake behaving mice via a closed and pressurized cranial window. We propose two specific aims to develop and apply this functional imaging approach to investigate factors that influence the activity of meningeal afferents in male and female mice under normal physiological conditions (locomotion vs. quiet waking) and during conditions linked to the development of headache and migraine. In Aim 1, we will test the hypothesis that, under normal physiological conditions, molecularly-defined meningeal afferent subpopulations serve as homeostatic sensors that detect rapid brain motion, changes in ICP and meningeal vascular responses related to locomotion. In Aim 2, we will use long-term two-photon imaging to compare the activity of the same meningeal afferents across sessions at baseline, during conditions that cause headache in humans, and following their resolution. Imaging combined with ex vivo molecular labeling will be used to define the molecular profile of afferents modulated by the headache triggers. The development of this innovative and powerful approach will provide a dramatically better understanding of the role of meningeal afferents in meningeal homeostasis and under pathophysiological conditions that lead to headache. Advances made using this approach could accelerate preclinical translational headache research.